A fluorescence microscope is an optical microscope that uses fluorescence and phosphorescence to generate an image. The specimen is illuminated with light of at least a specific wavelength, which is absorbed by the fluorophores, causing them to emit light of longer wavelengths. A typical fluorescence microscope includes a light source such as mercury arc lamp, an excitation filter, a dichroic beamsplitter, and an emission filter. The filters and the dichroic beamsplitter are chosen to match the spectral excitation and emission characteristics of the fluorophore used to label the specimen. Most fluorescence microscopes in use are epifluorescence microscopes, where excitation of the fluorophore and detection of the fluorescence are done through the same objective. These microscopes are widely used in biology and medical.
In order to acquire high-quality images in fluorescence microscopy systems, focusing is a crucial factor. Conventionally, microscopy focusing algorithms depend on the images themselves. A series of images at different focus planes is examined, and the image, having the largest amount of details, is selected as being correctly focused. For example, the focusing system takes a series of images around an estimated focus position. For each image, the system records its position, and computes a focus score which characterizes the sharpness of the respective image. Finally, the system computes a position for which the focus score is maximal, and the computed position is taken as the focus position.
U.S. Pat. No. 7,141,773 provides an imaging apparatus with an autofocus mechanism for obtaining focused images. The apparatus includes an objective lens, a focus controller for altering a distance between the objective lens and a sample, an object finder for finding objects of interest within the sample, and a light intensity measurement unit which measures light intensity levels of the thus identified objects of interest. The focus control alters the sample-objective distance to maximize the light intensity levels being measured, thereby to arrive at a focus position. Objects of interest may be identified by filtering out large objects and optionally by masking out background regions.
WO 2007138369 discloses a method and fluorescent imaging system for digitizing a specimen with fluorescent target points on a microscope slide. The method uses the fluorescence microscope system for detecting and scanning the fluorescent target points, and comprises the steps of marking the position of the specimen on the slide by visible marking means to define a specimen area containing the specimen and the fluorescent target points, capturing a bright field image of at least a portion of the slide at a first optical magnification, in which the portion contains the specimen area and determines a position of target fields falling within the specimen area from the visible marking means, and scanning the target fields of the specimen area at a second optical magnification higher than the first optical magnification, in which the scanning includes focusing on at least a part of the target fields.
The abovementioned prior arts are using the fluorescence-based focusing method, which enables focusing with high accuracy. Nevertheless, low fluorescence intensity requires long exposure to acquire an image with decent signal, so focus search based on multi-image comparison is slow. Furthermore, fluorescence intensity quickly decreases away from focus plane, thus making the focus search initiated far away from focus plane getting difficult. Long focusing time requires continuous excitation which may cause photobleaching. Slow focusing is particularly problematic for scanning a wide-field fluorescence microscopy. Ultimately, system throughput is getting low when too much time is taken for focusing.
For shortening the time for focusing and avoiding from photobleaching, non-fluorescence light is used for focusing. An additional light source such as halogen lamp is integrated with the fluorescence microscope for focusing. Mostly, other optical techniques such as phase contrast (PC) and differential interference contrast (DIC) are implemented as well in order to obtain high quality of focusing.
Phase contrast is an optical microscopy technique that converts phase shifts in light passing through a transparent specimen to brightness changes in the image. Phase shifts themselves are invisible, but become visible when shown as brightness variations. The basic principle to make phase changes visible in phase contrast microscopy is to separate the illuminating background light from the specimen scattered light, which makes up the foreground details, and to manipulate them differently. For performing such technique, special optics are employed like phase contrast annulus, condenser, and phase plate.
Differential interference contrast microscopy is also an optical microscopy illumination technique used to enhance the contrast in unstained, and transparent samples. DIC works on the principle of interferometry to gain information about the optical path length of the sample, to see otherwise invisible features. DIC works by separating a polarized light source into two orthogonally polarized mutually coherent parts which are spatially displaced (sheared) at the sample plane, and recombined before observation. Polarizer, DIC prism, condenser, analyzer are required for realizing the technique of DIC.
U.S. Pat. No. 5,790,710 teaches a phase-contrast autofocus for fluorescence microscopy.
U.S. Pat. No. 6,674,574 provides a focusing system for a microscope comprising an objective lens, a sample stage, a reflected illumination system for generating fluorescence from a sample, a transmitted illumination system for irradiating light on the sample to capture a transmitted optical image, a set of optical elements for forming the transmitted optical image on the basis of a phase information included in light transmitted through the sample, an optical element for dividing the fluorescence image and the transmitted optical image, a sensor for capturing the transmitted optical image divided by the optical element for dividing light, a focus detecting section for detecting a focusing level of the transmitted optical image on the basis of a signal output from the sensor, and a driver for moving at least one of the objective lens and the stage to focus on the sample on the basis of the focusing level.
Both U.S. Pat. No. 5,790,710 and U.S. Pat. No. 6,674,574 are using non-fluorescence based focusing method. They acquire an advantage in having fast speed in focusing. Nevertheless, their disadvantages are that only one focus is provided for all fluorescence channels, architecture of the microscope is complicate and much special optics are required.
Consequently, there is an unmet need for a focusing apparatus and method for fluorescence microscopy, especially wide-field scanning fluorescence microscopy applications like whole slide imaging and digital pathology, which provide fast speed in focusing under simple architecture and without employing too much special optics.